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1 . American Journal of Pathology:

1993 Feb, 142(2):403-412

1993, 143(4):1 150-1 158
1984, 114(3):454-460
1996, 148(3):865-875

1965 Sep, Vol. 44, pp. 280-282

2. Cancer Research, 1993 May 15, 53(10 suppl):2287-2299

3. Cancer epidemiology, biomarkers and Prevention, 1996 Jul, 5(7):549-557

<5f4~. Lab Invesiigatjen

^T98 0,42(1):91-96^ >
iy88, 58(2): 141 -149

5. Gynecol Oncol, 1982, 13(1):58-66

6. International Journal of Gynecological Pathology:

1985,4(4):300-313
1986, 5(2):151-162
1992, 11(1):24-29

7. Differentiation:

1986, 31 (3): 191 -205
1988, 39(3): 185-1 96

8. Cancer (Phila), 1989, 63(7): 1337-1 342

9. Cancer Res, 1990, 50(16):5143-5152

10. Virchows Arch B Cell Pathol Incl Mol Pathol, 1987, 54 (2):98-1 10

1 1 . Acta Histochemica et Cytochemica:

1994, 27(3):251-257
1996, 29(1):51-56

12. Archives of Gynecology and Obstetrics, 1989, 246(4):233-242

13. Clin Lab Med, 1995 Sep, 15(3):727-742

14. Clin Obstet Gynaecol, 1 984 Apr, 1 1 (1 ):5-23

l

0023^837/80/4201-0091302.00/0
Laboratory Investigation

Copyright © 1980 by the United Stat^Canadian Division of the International Academy of Pathology Vol 42, No. 1. p. 91,1980

Printed in U.S.A.

Immunohistochemical Localization of Keratin
in Normal Human Tissues

Richard Schlecel, M.D., Ph.D., Susan Banks-Schlecel, Ph D and

Geraldine S. Pinkus, M.D. • "<•"•. and

lechnology, Cambridge, Massachusetts 1

Immunohistochemical identification of intracellular k» ra «>„ .„ .. .
antibody technique on paraffm^mbedded huma^ tis^ A ^t J * chle ™* «">ing an indirect
keratins are abundant in aU layers of snu„m™,^»t t- ? f ""nerous tissues confirms that

and in the epithelia of ^nJS^TEu^SSS U^fT of ^^^ed glands,*
which offers increased histologic resolution" w^ve .fho Ji ^!^ """"•""Peroxidase technique
tracheal, bronchial, prostatic, and cereal did eSfteU-^L ** J""? W T^ 6 ™ of «"
ceUs in these tissues. The nornial d^^Slt^ U c ^^^^ keratin-containing
cells is accompanied by the loss of cytooWmic k^ n ^ ™ * nondividing, superficial columnar
metaplasia stain intensely MithSitikeStiS MtitSJteJ aSl ESSi? 0- ' ° f epitheUal famous
proliferation of the keratin-containinTbS wll^^ "W* an ex ***e r *«*

various glands, and many mesodermal ti^e^rn. ^^ ? 8pi f a ^ ry eP'thellum, acinar cells of
and connects tissue) wer^mXfaS^ MMtte ' nerve,

within fixed, embedded tissue (uStadfag ££e ES^^K&i* 0 5f ratfal protehlfl

useful fa the study of tissue histogenesff SZZESSL^ tJS^IJtW^ may P rove
Poorly differentiated malignant neoplasms andTmor^ of

no^SdaisSSg^ 1 Kerat,D Pr ° telB8 ' EpitheBal ^""tiation, Squamous metaplasia, Immu-

ticipate in the in vitro assembly ofTnn^ tente (2« taffSE? ^ ^ T - an ^P^lial local-

and antibodies prepared against a sSsSX ^ , i tl " inhuman lung and in glands which is

ular weight epidermal pro^ve^n sho^S" Z^^™?^ ** described » *e rabbit. In

ize ultrastructuraUy to the 8-nm. uSaS^idS iffSf' tb " I »*oil ataoU prove to be useful in the

dermis (5, 12). ronoiuamente of epi- pathologic analysis of poorly differentiated human tu-

Only recently has it been recognized that keratin fila-

^W«itW,Wia MATERIALS AND METHODS

that in vitro cultures of epithelia such as human kidney Antigen Preparation

pattern. When frozen Sn^rfS <Jamentous of Sun and Green (28). Stratum corneum (600 mg)
stained with the^Te SSy^flu^n^T

te<^ fa the myoepitheKum ^ wtun^uT! ^y^^hyOaimnometitahe

and in the biliary duct cells of the nW^W^n^nd I P " 7A ' ™ tU tis8ue Was dis-

Green (27) andSun.Shih, and Grin?2^u^ed kerati^ TlTXn" f - 8us Pf nslon wa ^ centrifuged at 12.000 r.pan.

distribution in various rabbit aa^SiXSS ^ HZ' 300 ? e) for 20 nunute8 m a SorvaI » RC2-B centrifuge,

unmunofluorescence ^ ™ e ^f was extracted twice with 20 ma. Tris-HcfpH

™««opy ana clearly defined 7.4, and three times with 20 nut Tris-HCl, pH 7.4, con-

92

SCHLEGBL, BANKS-SCHLEGEL, AND PINKUS

Laboratoby Investigation

taining 8 M urea to remove water-soluble proteins, leaving
the water-insoluble keratin in the pellet. The insoluble
peUet was then extracted with 20 mat Tris-HCl, pH 7.4,
containing 8 M urea and 10 mM dithiothreitol (to solubil-
ize the disulfide cross-linked protein), sonicated for 15
seconds at a setting of 5 on a Bronson sonifier and
centrifiiged. The supernatant fluid was used as antigen.

POLYACRYLAMIDE GEL ELECTROPHORESIS

Purified keratin protein (30 fig.) was applied to a 10
per cent polyacrylamide gel with a stacker gel of 5 per
cent polyacrylamide. Both gels contained 0.1 per cent
sodium dodecyl sulfate, essentially as described by La-
emmli (15). Electrophoresis was performed at 30 ma.
until the tracking dye reached the bottom of the gel (4
hours). After staining with Coomassie blue dye, keratin
proteins varying from 41,000 to 65,000 daltons could be
identified (Fig. 1). Similar patterns have been obtained
by Sun and Green (28).

Antibody Preparation

Keratin proteins purified from human stratum cor-
neum as described above were emulsified with Freud's
complete adjuvant and injected intradermally into New
Zealand White rabbits, using methods described previ-
ously (28). One milligram of keratin protein was used for
the primary injection. One month later, a second injec-
tion containing 0.5 mg. of protein was given. Serum was

A B

Fig. 1. Sodium dodecyl sulfate gel electrophoresis pattern of keratin
proteins selectively extracted from human stratum corneum. Molecular
weight standards on the left side of the gel are: bovine serum albumin
(68,000). tubulin (57,000 and 55,000), actin (42,000), aldolase (40,000),
chymotrypsinogen (26,000), myoglobin (17,000), and cytochrome c
(12,000).

collected 7 to 10 days after the second injection. Double
diffusion studies in 1 per cent agarose gels containing 0.1
per cent sodium dodecyl sulfate and 0.5 per cent Triton
X-100 (30) revealed a single, strong precipitin band be-
tween the antiserum and keratin proteins extracted from
stratum corneum. Preabsorption of the antiserum with
keratin proteins eliminated this band; 0.6 ml. of antise-
rum was preabsorbed by overnight incubation at 37° C.
with keratin extracted from 0.2 gm. of human stratum
corneum. There was no immunologic cross-reactivity of
immune or preimmune serum with proteins of cultured
3T3 fibroblasts or fibroblasts derived from human skin.
Prior to use in immunoperoxidase studies, the preim-
mune and immune sera were adsorbed with mouse liver
powder to minimize nonspecific staining.

Immunoperoxidase Staining

Paraffin-embedded tissues were retrieved from the sur-
gical pathology files of the Peter Bent Brigham Hospital,
and immunoperoxidase staining of these tissues was per-
formed according to a modification (21) of published
methods (16, 18, 26). Unless otherwise specified, all tis-
sues were fixed in 10 per cent buffered formalin. Sections
(5 pm. thick) were transferred to glass slides and warmed
to 45° C. for 1 hour to ensure adherence. The sections
were deparaffinized in xylene, placed in absolute alcohol,
then sequentially incubated for 30 minutes at room tem-
perature with each of the following reagents: (1) metha-
nolic hydrogen peroxide (1 volume of 3 per cent aqueous
hydrogen peroxide to 5 volumes of methanol); (2) rabbit
antihuman keratin antiserum (1:20 to 1:100 dilution); (3)
swine antirabbit serum IgG (1:20 dilution); (4) horserad-
ish peroxidase-rabbit antihorseradish peroxidase-soluble
complexes (1:100 dilution). All dilutions were performed
with 0.1 m Tris-buffer, pH 7.6. To decrease nonspecific
background staining, sections were incubated with either
5 per cent egg albumin or normal swine serum (1:10
dilution) for 30 minutes following methanolic peroxide
treatment. After each incubation, slides were washed
with Tris-saline (1 volume Tris buffer, 0.1 M, pH 7.6, to
9 volumes normal saline), then placed in Tris buffer for
15 minutes. Antibody localization was determined by
detection of peroxidase activity, effected by a 5-minute
incubation of the slides with a freshly prepared solution
containing 3,3'-diaminobenzidine tetrahydrochloride
(Sigma Chemical Company, St. Louis, Missouri, 6 mg.
per 10 ml. of Tris buffer) and 0.1 ml. of 3 per cent
hydrogen peroxide. This method yields a brown reaction
product. Slides were then washed with water, counter-
stained with hematoxylin, dehydrated, and mounted in
Permount.

Horseradish peroxidase-rabbit antihorseradish peroxi-
dase-soluble complexes and swine antirabbit serum IgG
were purchased from Dakopatts A/S of Copenhagen,
Denmark (United States agent, Accurate Chemical and
Scientific Company, Hicksville, New York).

RESULTS

In Table 1 is defined the distribution of keratin proteins
in a wide variety of adult human tissues. For all tissue
with detectable intracellular keratin proteins, the speci-

Vol. 42, No. 1,1980

KKRATIN PK0TKIN8 IN HUMAN TI8SUKS

Table I. Distribution of Intracvtoplasmic Keratin Proteins
in Normal Human Tissues as Determined by
Immunoperoxidase Studies

TixsueA

Result

Tbwue*

Kewjlt

Skin

Epidermis +

Hair follicle +

Sebaceous gland
Sebaceous cell ( + )"

Basal cell +

Sweat gland
Ductal cells +

Dermal collagen -
Lung

Trachea and bronchus
Columnar epithe- -
Hum

Basal cells +
Intermediate cells +
Submucosal glands
Ducts +
Acini —
Alveoli —
Cervix
Rxocervix
Squamous epithe- +
lium
Endocervix
Columnar epithe- -
lium

Basal cells +
Vagina

Squamous epithelium +

Prostate
Columnar cells -
Basal cells +

Oral cavity
Tongue

Squamous mucosa +
Buccal mucosa +

Parotid gland
Ducts +
Acini " -

Submaxillary gland
Ducts +
Acini —

Pancreas

Ducts

Acini

Islets
Breast

Urinary bladder and
ureter

Transitional epithe-
lium

Smooth muscle
Kidney

Glomeruli

Tubules, collecting

Tubules, proximal and
distal
Liver

Hepatocytes

Bile ducts

Hepatic arteries, veins
Testis

Tubules

Interstitium
Adrenal
Ovary
Stomach
Intestine

Ileum

Colon
Nerve
Muscle

Smooth

Skeletal
Connective tissue
Lymph node
Spleen

Hematopoietic

(iliac crest)

Erythroid, myeloid,
and megakary-
ocyte lines

Bone trabeculae

" Weak staining,

* Variable positivity in epithelial and myoepithelial cells of ducts and
acinar epithelium (see text).

' Weak staining was observed in these tissues with immune, and with
preimmune or keratin-absorbed serum and was therefore interpreted
as nonspecific.

ficity of the staining was verified by parallel experiments
using both neutralized antiserum and preimmune rabbit
serum.

Keratin proteins were detected equally in all layers of
the epidermis from stratum basale to stratum corneum,
and in some skin adnexa including hair follicles, sweat
gland ducts, and ducts of sebaceous glands (Fig. 2A).
These observations are consistent with the findings of
Sun and Green (28), except for the staining characteris-
tics of the stratum granulosum. In the latter study,
immunofluorescence with antikeratin antibodies dem-
onstrated little fluorescence in the granular layer, pre-

83

sumably due to a quenching phenomenon. In addition,
preimmune serum reacted weakly, but specifically, with
this same layer. Immunoperoxidase staining exhibited
neither of these characteristics. The stratum granulosum
reacted as well with antikeratin antibodies as did the
other layers, and none of the layers stained when using
either preimmune or keratin-absorbed antiserum (Fie
2B). '*

Other squamous epithelia such as oral mucosa, exocer-
vix, and vaginal mucosa also stained strongly, although
an unequal distribution of staining was found in the
female genital tract. Whereas the basal epithelial cells of
vaginal mucosa stained strongly, the upper layers (which
contain variable amounts of glycogen in response to
estrogen stimulation) reacted only weakly with the anti-
serum.

Epithelial-derived glands such as parotid gland (Fig.
2D), submaxillary gland, and pancreas (Fig. 2H), revealed
relatively similar keratin distributions. The ductal epi-
thelial cells of each of these glands, which are single
layered, cuboidal, or columnar cells, contained significant
amounts of intracellular keratin, whereas the acinar
structures were uniformly negative. Staining within the
parotid ductal system extended from the large striated
ducts to the small intercalated ducts, despite the lack of
detectable tonofilament bundles in these cells by electron
microscopy (20, 23). Foci of squamous metaplasia in such
ductal systems (e.g., Fig. 2H) f represented a focal prolif-
eration of celt which stained intensely for keratin.

A different staining pattern was noted in the bronchial
epithelium of the lung (Fig. 2Q, cervix (Fig. 2E) t and
prostate (Fig. 2F). In general, these tissues contain an
epithelium which is composed of superficial columnar
cells and underlying basal or reserve cells. In the lung,
the basal cells (and intermediate cells) contained signifi-
cant amounts of keratin, unlike the ciliated and mucus
columnar cells which lacked this protein. The cervix and
prostate are bilayered, and only the basal cells located
adjacent to the basement membrane contained ke vein.
Again, the superficial columnar cells were negative.

Squamous metaplasia is common in the bronchial and
cervical epithelium and is believed to be related to the
development of squamous cell carcinoma (2). In both of
these tissues the appearance of squamous metaplasia
appears to be due to the focal proliferation of the basal,
keratin-positive epithelium. Areas of squamous metapla-
sia, regardless of tissue origin, stained strongly with an-
tikeratin antibodies.

The staining characteristics of the breast were variable
and dependent upon the specific fixative used. For ex-
ample, formalin-fixed breast tissue displayed homogene-
ous staining of the entire ductal epithelium (consisting of
the myoepithelium and the superficial ductal cells). In
contrast, breast tissue fixed with B5 solution (4) shows
an augmentation of myoepithelial cell staining relative to
the overlying ductal cells (Fig. 2G). The latter findings
are comparable to those described in previous immuno-
fluorescence studies (8). All other tissues examined in
this study had characteristic staining patterns which
were independent of the fixation procedure.

As described for th- rabbit (29), keratin was present in

94

SCHLRGEI„ BANKS-SCHLBGEU AND PINKUS

Laboratory Investigation

Fig. 2. Inununohistochemical localization of intracellular keratin in
paraffin sections of normal human tissues. A, Skin. Keratin proteins
(brown) present in all layers of epidermis, fi. Skin. Absorption of
immune serum with keratin protein completely abolishes staining re-
action. C, Bronchus. Strong staining for keratin proteins in basal and
intermediate cells. Ciliated and mucus columnar cells are devoid of
detectable keratin proteins. D, Parotid. Keratin proteins identified in
ductal epithelium. Acini are negative. E, Endocervix. Basal cells of
endocervical glands demonstrate strong staining for keratin proteins.

Mucus columnar cells are negative. F, Prostate. Staining for keratin
proteins observed in basal cells of prostatic glands. Columnar cells are
negative. G t Breast (B5 fixative). Prominent staining for keratin pro-
teins in myoepithelial layer of duct, with weaker reaction in superficial
epithelial cells. H, Pancreas. Staining for keratin proteins observed in
ductal epithelium with increased staining intensity in areas of squamous
metaplasia. Acini are negative. All tissues were counterstained with
hematoxylin. Figure 2A to C, X250; D and H, X125; £, X500; Fand G,
X250.

Vol.42, No. 1, 1980

KERATIN PROTEINS IN HUMAN TISSUES

95

the urothelium of the bladder and ureter and extended
into the collecting duct system of the kidney. Keratin
was absent in renal glomeruli. In the liver, only the bile
ducts contained detectable keratin protein.

Staining characteristics of the gastrointestinal tract
were equivocal. While there was a minimal degree of
reactivity of such epithelia with the antikeratin anti-
bodies, the staining was not abolished by the absorption
of the antiserum with keratin proteins, and weak staining
occurred even with the use of preimmune serum. We
have interpreted these results as representing a nonspe-
cific reaction.

DISCUSSION

The cytoplasm of mammalian cells contains a complex
array of filamentous proteins which participate variously
in the maintenance of cell shape and mobility (6), mod-
ulation of cell membrane protein movement (1, 19), mo-
bility of chromosomes and cellular processes (10), cyto-
kinesis (24), and possibly the regulation of cell prolifera-
tion (7). Keratin filaments, which are one type of cyto-
plasmic "intermediate" filament, are found principally in
epidermal cells where they are believed to have a struc-
tural function. The recent immunologic detection of ker-
atin proteins in many types of epithelia suggests that
these filaments have a more diverse biologic distribution
than previously recognized (8, 9, 27, 29). In addition, the
strong reactivity of parotid ductal epithelium with anti-
keratin antiserum (Fig. 2D) and the absence of tonofila-
ment bundles by electron microscopy (20) indicate that
these epithelial cells contain abundant keratin proteins
which are present in a nonaggregated form, ultrastruc-
turally indistinguishable from other intermediate-sized
filaments.

The present investigation represents the first compre-
hensive study to localize keratin proteins in human tissue
and to identify specifically the basal cells of trachea,
bronchi, prostate, and cervix as the predominant sites of
keratin synthesis. Bronchial basal cells, which have been
shown to contain numerous tonofilament bundles by
electron microscopy (23), apparently represent the pro-
liferating cells of the respiratory epithelium (3, 13). It is
interesting to note that, during the differentiation of
these basal cells into nondividing columnar cells, there is
a concomitant loss of cellular keratin proteins by a mech-
anism not yet known. The histology of the normal human
bronchus has recently been reviewed and examined by
McDowell et al (17). In this study, the cells commonly
referred to by histologists as "intermediate" were shown
to consist of a mixture of basal cells and small mucus
granule cells which had similar appearances ultrastruc-
turally, except that the small mucus granule cells often
contained small, cytoplasmic periodic acid-Schiff-posi-
tive granules. Both cell types were considered to have a
proliferative potential, based upon extrapolation from
hamster experiments. Our studies demonstrate that both
basal and intermediate cells (presumably including small
mucus granule cells) contain prominent intracellular ker-
atin protein. The recent finding that the analogous ker-
atin-positive cells of the cervix also have a proliferative
capacity (11) suggests that the basal cells of the prostate

and breast may fulfill similar precursor-cell roles. The
basal or reserve cells of the lung and cervix can proliferate
in either a regulated fashion to produce regions of squa-
mous cell metaplasia, or in an unregulated manner to
produce squamous cell carcinoma (14).

Immunofluorescence studies with rabbit tissue have
demonstrated the presence of keratin in both superficial
and basal layers of trachea, bronchi, and cervical glands
(29). Whether the difference between the rabbit and
human tissues reflects species-related variation in gland
architecture or keratin localization, or whether it results
from differences in the sensitivity or resolution of the
particular immunologic technique is unclear.

Keratin has a similar distribution pattern in the sali-
vary glands, pancreas, and lung. These tissues arise de-
velopmentally by the ingrowth and sequential branching
of an epithelial growth bud and, in each case, the keratin-
containing cells are found only in the early branchings,
that is, the ducts or bronchi. The terminal acini or alveoli
lack keratin protein. Presumably, the process of gland or
organ development results in the loss of keratin synthesis.

The detection of tonofilament bundles (aggregates of
keratin filaments) by electron microscopy has been used
for distinguishing poorly differentiated carcinomas from
sarcomas and lymphomas. However, immunoperoxidase
staining for keratin protein has significant advantages
over electron microscopy for defining the epithelial na-
ture of a tumor a greater number of cells may be exam-
ined histologically; special fixation and processing are not
necessary; the technique is rapid; and, most importantly,
the keratin filaments need not necessarily be aggregated
into characteristic bundles for their conclusive identifi-
cation. Also, this immunohistochemical method affords
excellent cellular detail and a permanent preparation.
Attempts to extend the use of this technique to the
differential diagnosis of benign and malignant human
neoplasms as well as to the various "undifferentiated"
tumors are in progress.

Acknowledgments: We thank Dr. T. T. Sun for helpful
suggestions and comments and for communicating the
results of his keratin localization studies while they were
in progress. We also thank Dr. H. Green and Dr. Ramzi
S. Cotran for reviewing the manuscript. The expert tech-
nical assistance of Ms. Janet A. McLeod and Ms. Barbara
Baron and the skilled secretarial assistance of Ms. Ann
Benoit and Ms. Margaret Cialdea was greatly appreci-
ated.

Date of acceptance: October 10, 1979

This work was supported in part by the Milton Fund of Harvard
University and Grant HL-06370 from the National Institutes of Health.

Address reprint requests to: Dr. Geraldine S. Pinkus, Department of
Pathology. Peter Bent Brigham Hospital, 721 Huntington Avenue,
Boston, Massachusetts 021 15.

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96

SCHLEGEL, BANKS-SCHLEGEL. AND PINKUS

Laboratory Investigation

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